Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
PRAME Purification
TECHNICAL FIELD
The present invention relates to methods for the purification of PRAME.
BACKGROUND
PReferentially expressed Antigen in MElanoma", or "PRAME", is a tumour
antigen encoded by the PRAME gene.
PRAME is an antigen that is over-expressed in many types of tumours,
including melanoma, lung cancer and leukaemia (Ikeda et al., Immunity 1997, 6
(2)
199-208). A high level of PRAME expression has been reported for several solid
tumors, including ovarian cancer, breast cancer, lung cancer and melanomas,
medulloblastoma, sarcomas, head and neck cancers, neuroblastoma, renal cancer,
and Wilms' tumour and in hematologic malignancies including acute
lymphoblastic
and myelogenous leukemias (ALL and AML), chronic myelogenous leukemia (CML),
Hodgkin's disease, multiple myeloma, chronic lymphocytic leukemia (CLL) and
mantle cell lymphoma (MCL).
PRAME is also expressed at a very low level in a few normal tissues, for
example testis, adrenals, ovary and endometrium.
PRAME represents an important anti-cancer immunotherapeutic. In
immunotherapy the cancer antigen is introduced to the patient usually as a
vaccine,
for example containing the protein or an antigenic fragment thereof, which
stimulates
the patient's immune system to kill tumours expressing the same antigen.
The production of a vaccine comprising the cancer antigen, in this case PRAME,
requires a significant quantity of the cancer antigen, which in turn calls for
the large
scale expression and purification of the antigen.
SUMMARY OF THE INVENTION
PRAME is over expressed in E.coli where it forms inclusion bodies. In order to
solubilise PRAME from the inclusion bodies they must be exposed to strongly
solubilising conditions requiring anionic detergent and urea. However, such
conditions are not suitable for the final formulation of PRAME into a
composition for
injection into patients and the purified PRAME must be transferred to another
diluent.
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The inventors of the present application have realised that transfer of PRAME
from a
diluent comprising the anionic detergent used to solubilise it to one which is
substantially free of that anionic detergent causes aggregation of PRAME. This
aggregation continues over time and eventually causes precipitation of the
PRAME
out of solution. As this aggregation (antigen size evolution) is not suitable
for use in
an immunotherapeutic composition there is therefore a need in the art for
improved
methods for the purification of PRAME.
Methods and processes for reducing the aggregation of PRAME, as well as
compounds produced by these methods and processes, are provided herein. In one
embodiment there is provided a method for reducing the aggregation of a
protein
during a diluent exchange from diluent A to diluent B comprising: (i) adding a
polyanionic compound to diluent A prior to the exchange; and (ii) exchanging
the
protein from diluent A to diluent B, wherein the protein is PRAME. In one
embodiment there is provided the use of a polyanionic composition for reducing
the
aggregation of a protein during a diluent exchange from diluent A to diluent
B,
wherein the protein is PRAME. In one embodiment the polyanionic compound is
added prior to the diluent exchange. In one embodiment diluent A comprises a
detergent. In another embodiment the detergent is an anionic detergent. In
another
embodiment the detergent is selected from the group consisting of: SDS, sodium
docusate and lauryl sarcosyl.
In one embodiment diluent B is substantially free of detergent.
In one embodiment the polyanionic compound is an oligonucleotide. In one
embodiment the oligonucleotide is 5 to 200 nucleotides in length. In one
embodiment, the oligonucleotide comprises a CpG. Most In one embodiment the
oligonucleotide is selected from the group consisting of: TCC ATG ACG TTC CTG
ACG TT (CpG 1826) (SEQ ID NO:1); TCT CCC AGC GTG CGC CAT (CpG 1758)
(SEQ ID NO:2); ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG (SEQ ID
NO:3); TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006/CpG7909) (SEQ ID
NO:4); TCC ATG ACG TTC CTG ATG CT (CpG 1668) (SEQ ID NO:5); or TCG ACG
TTT TCG GCG CGC GCC G (CpG 5456) (SEQ ID NO:6).
In one embodiment, the diluent exchange is achieved by dialysis, diafiltration
or size exclusion chromatography.
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In one embodiment, the method further comprises step (iii) formulating the
protein into diluent C. In one embodiment diluent C comprises Tris, Borate,
sucrose,
poloxamer and CpG.
The invention also provides a composition comprising PRAME in diluent C as
produced by the methods of the invention.
The invention also provides a composition comprising PRAME and an
oligonucleotide, wherein the PRAME has a particle size of between 10-30nm. In
another embodiment PRAME has a particle size of between 15-25nm. In another
embodiment, the oligonucleotide comprises a CpG. In a further embodiment, the
particle size is determined by dynamic light scattering.
The invention also provides a method of producing a pharmaceutically
acceptable PRAME composition comprising the steps of: (a) carrying out a
diluent
exchange according to the methods of the invention; (b) formulating the
protein into
diluent C; and (c) sterilising the formulation produced in step (b). In
another
embodiment, the method comprises the additional step (d) lyophilising the
formulation produced in step (c). In another embodiment, step (c) is achieved
by
filtration.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1/21: Electrophoretic mobility measurement and Zeta potential
calculation
for PRAME purified antigen with Malvern ZetaSizer Nano ZS equipment.
Figure 2/21: Light scattering (LS), refractive index (RI) and molar mass (MM)
distributions as determined by SEC-MALLS analyses for GMP lot DPRAAPA003 at
release.
Figure 3/21: Light scattering (LS), refractive index (RI) and molar mass (MM)
distributions as determined by SEC-MALLS analyses for GMP lot DPRAAPA004 at
release.
Figure 4/21: Light scattering (LS), refractive index (RI) and molar mass (MM)
distributions as determined by SEC-MALLS analyses for GMP lot DPRAAPA005 at
release.
Figure 5/21: Sedimentation coefficient distributions c(s) obtained by SV-AUC
analyses of GMP lots DPRAAPA003 (blue profile), DPRAAPA004 (red profile) and
DPRAAPA005 (green profile) at release. Note that the raw data obtained by SV-
AUC
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have been processed using the Sedfit software. The waves are due to this
signal
treatment and, thus, are artifactual.
Figure 6/21: SDS-PAGE analysis in reducing conditions on 4-12% Bis-Tris
polyacrylamide gel Coomassie Blue R250 staining (5 pg of protein loaded per
lane) -
Final Container reconstituted in ASA (Sorbitol) - Follow-up of the
reconstitution
kinetic at 25 C. Lanes are numbered from left to right
Figure 7/21: Western Blot analysis against PRAME antigen. Final Container
reconstituted in ASA (Sorbitol) buffer or water. Follow-up of the
reconstitution kinetic
at 25 C. 0.3pg of protein loaded per lane, transfer on nitrocellulose membrane
1h at
100V, alkaline phosphatase (NBT-BCIP) detection. Lane 1: Final container (FC)
reconstituted in water for injection at TO ¨ non centrifuged sample; lane 2:
idem 1 ¨
centrifuged sample (supernatant); lane 3: Final container (FC) reconstituted
in ASA
buffer at TO ¨ non centrifuged sample; lane 4: idem 3 ¨ centrifuged sample
(supernatant); lane 5: Final container (FC) reconstituted in ASA buffer at T
4h 25 C ¨
non centrifuged sample; lane 6: idem 5 ¨ centrifuged sample (supernatant);
lane 7:
Final container (FC) reconstituted in ASA buffer at T24h 25 C ¨ non
centrifuged
sample; lane 8: idem 7 ¨centrifuged sample (supernatant).
Figure 8/21: Isothermal titration calorimetry profile corresponding to the
stepwise
injection of CpG7909 into a PRAME solution. Binding of CpG to PRAME results in
the characteristic sequence of the signal, until saturation is reached.
Figure 9/21: Top panel represents the PRAME protein distribution visualized
after
silver staining of a SDS-PAGE gel. Bottom panel represents the CpG
distribution
along the gradient after IEX-HPLC-UV determination. Fraction 1 is equivalent
to the
bottom fraction highlighted above the corresponding lane of the SDS-PAGE gel.
Similarly, fraction 12 is equivalent to the top fraction and fraction w is
equivalent to
the tube wash lane. Red box is meant to delineate the fractions were CpG is
interacting with the antigen (in control experiment, CpG alone is found in top
fractions only).
Figure 10/21: Comparative data showing the amount of CpG associated with
PRAME antigen for three distinct repro lots. Blue bars correspond to ex-tempo
reconstitution of lyophilized materials (500 pg dose on left half of graph,
100 pg dose
for right half). Green bars correspond to samples pre-incubated for 24h at 25
C
before ultracentrifugation. Diamond-shaped in magenta correspond to the mass
ratio
CpG/Ag and should be read from the right axis.
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Figure 11/21: SEC-HPLC method development. SEC Column selection. UV profiles
obtained on different TSK columns for purified antigen.
Figure 12/21: SEC-HPLC method development. SEC Column selection. UV profiles
obtained on different TSK columns for purified antigen spiked with CpG
solution
(1050 pg/ml).
Figure 13/21: SEC-HPLC analysis on TSK G4000 PWx1 + G6000 PWx1 columns (+
guard column) equilibrated in 5 mM Borate buffer pH 9.8 ¨ 3.15% sucrose (=
buffer
of the purified antigen) at a flow-rate of 0.5 ml/min and with UV detection at
220 nm ¨
UV profiles obtained for purified antigen alone or after spiking with
increasing
concentrations of CpG. CpG impact on antigen chromatographic profile. N.B. Vo
=
void volume of the column, i.e. the volume outside of the resin beads.
Figure 14/21: SEC-HPLC analysis on TSK G4000 PWx1 + G6000 PWx1 columns (+
guard column) equilibrated in 5 mM Borate buffer pH 9.8 ¨ 3.15% sucrose (=
buffer
of the purified antigen) at a flow-rate of 0.5 ml/min and with UV detection at
220 nm ¨
UV profiles obtained for CpG solution in water from 10 pg/ml up to 1050 pg/ml.
Figure 15/21: Size analysis by dynamic light scattering ( ZetaNano from
Malvern)
on purified antigen samples spiked or not with excipient and stored 24h at 22
C (no
size measurement done when antigen precipitation observed by visual
observation).
Figure 16/21: Size analysis by dynamic light scattering (ZetaNano from
Malvern)
on purified antigen samples spiked with selected excipient candidates and
stored 14
days at +4 C.
Figure 17/21: Turbidity measurement (HACH 2100AN IS(D) on purified antigen
samples spiked with selected excipient candidates after 14 days at +4 C.
Figure 18/21: Compatibility of ASA (Sorbitol) with ionic detergents - Size
analysis by
dynamic light scattering (ZetaNano from Malvern).
Figure 19/21: A graphical representation of DLS measurements.
Figure 20/21: Visual analysis of a samples with no CpG (R19/1) and spiking
with
100pg/m1CpG in HA-FT prior UF (Run R26/1).
Figure 21/21: A graphical representation of DLS measurements.
DETAILED DESCRIPTION
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The inventors have surprisingly found that the addition of a polyanionic
compound to a diluent containing PRAME prior to the exchange of the diluent
reduces the aggregation of PRAME.
Aggregation
As discussed above, the methods of the invention reduce the aggregation of
PRAME during a diluent exchange. Aggregation refers to the associating of
individual PRAME molecules with other PRAME molecules to form multimers.
Aggregation can be observed visually or using dynamic light scattering
techniques
well known in the art.
PRAME
As described above, PRAME is an antigen that is over-expressed in many
types of tumours, including melanoma, lung cancer and leukaemia (Ikeda et al.,
Immunity 1997, 6 (2) 199-208). The PRAME protein has 509 amino acids (SEQ ID
NO:7). The antigen is described in US patent No. 5, 830, 753. PRAME is also
found
in the Annotated Human Gene Database H-Inv DB under the accession numbers:
U65011.1, BCO22008.1, AK129783.1, BC014974.2, CR608334.1, AF025440.1,
CR591755.1, BC039731.1, CR623010.1, CR611321.1, CR618501.1, CR604772.1,
CR456549.1, and CR620272.1. As used herein, the term PRAME includes the full
length wild type PRAME protein. It also includes PRAME proteins with
conservative
substitutions. In one embodiment, one or more amino acids may be substituted,
i.e.
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more. The
PRAME
protein may additionally or alternatively contain deletions or insertions
within the
amino acid sequence when compared to the wild-type PRAME sequence. In one
embodiment, one or more amino acids may be inserted or deleted, i.e. 2, 3, 4,
5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more.
In one embodiment, the term PRAME includes proteins which share 80% or
more sequence identity with the full length wild type PRAME protein, i.e.,
85%, 90%,
95%, 96%, 97%, 98%, 99% or more.
The term PRAME also includes fusion protein proteins comprising the
PRAME protein. PRAME may be fused or conjugated to a fusion partner or carrier
protein. For example, the fusion partner or carrier protein may be selected
from
protein D, NS1 or CLytA or fragments thereof. See, e.g., W02008/087102.
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In one embodiment of the invention, the immunological fusion partner that may
be
used is derived from protein D, a surface protein of the gram-negative
bacterium,
Haemophilus influenza B (W091/18926) or a derivative thereof. The protein D
derivative may comprise the first 1/3 of the protein, or approximately the
first 1/3 of
the protein. In one embodiment, the first 109 residues of protein D may be
used as a
fusion partner. In an alternative embodiment, the protein D derivative may
comprise
the first N-terminal 100-110 amino acids or about or approximately the first N-
terminal 100-110 amino acids. In one embodiment, the protein D or derivative
thereof may be lipidated and lipoprotein D may be used.
In one embodiment, the PRAME protein is a fusion protein comprising: a)
PRAME or an immunogenic fragment thereof, and b) a heterologous fusion partner
derived from protein D, wherein the said fusion protein does not include the
secretion
sequence (signal sequence) of protein D. By secretion or signal sequence or
secretion signal of protein D is meant the N-terminal 19 amino acids of
protein D.
Thus, the fusion partner protein of the present invention may comprise the
remaining
full length protein D protein, or may comprise approximately the remaining N-
terminal third of protein D. For example, the remaining N-terminal third of
protein D
may comprise approximately or about amino acids 20 to 127 of protein D. In one
embodiment, the protein D sequence comprises N-terminal amino acids 20 to 127
of
protein D.
In one embodiment, the PRAME may be Protein D-PRAME/His, a fusion
protein comprising from N-terminal to C-terminal: amino acids Met-Asp-Pro;
amino
acids 20 to 127 of Protein D; PRAME; an optional linker; and a polyhistidine
tail
(His). Examples of linkers and polyhistidine tails that may optionally be used
include
for example: TSGHHHHHH; LEHHHHHH or HHHHHH.
PRAME as used in the present invention will usually be at a concentration
between 10-2000mg/ml, i.e. 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125,
150,
175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,
900, 950,
1000, 1100, 1200, 1300, 1400, 1500, 1750 or 2000mg/ml.
Polyelectrolytes and Polyanionic Compounds
Polyelectrolytes are polymers whose repeating units bear an electrolyte
group. These groups will dissociate in aqueous solutions (water), making the
polymers charged. Polyelectrolyte properties are thus similar to both
electrolytes
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(salts) and polymers (high molecular weight compounds), and are sometimes
called
polysalts. Like salts, their solutions are electrically conductive. Like
polymers, their
solutions are often viscous.
As referred to herein, a polyanionic compound is a polyelectrolyte with an
overall negative charge. Examples of polyanionic compounds include, but are
not
limited to, PLC and oligonucleotides.
The net negative charge at pH7.0 of the polyanionic compound may be
calculated by any suitable means. This may be an average property of the
compound, and should be calculated with respect to the Mw of the polyanionic
compound used. For instance, a PLC polymer with on average 17 residues should
have a net negative charge of 17. In one embodiment, the net negative charge
should be at least 8, or at least 17, preferably between 8-100, 10-80, 12-60,
14-40,
16-20, and most preferably about or exactly 17.
In one embodiment the polyanionic compound of the invention has at least
one average 1 net negative charge at pH 7.0 per 3 monomers, preferably at
least 2
per 3 monomers, and most preferably at least on average 1 net negative charge
for
each 30 monomer. The charges may be unevenly arranged over the compound
length, but are preferably evenly spread over the compound length.
The skilled person will appreciate that the term polyanionic compound may
include polyanionic detergents. However where the invention refers to adding a
polyanionic compound to diluent A prior to a diluent exchange from diluent A
to
diluent B, wherein diluent A comprises an anionic detergent, then the anionic
detergent is not the same as the polyanionic compound added to diluent A.
Poly L-glutamate (PLG)
Poly L-glutamate is a polymer of l-glutamate used to stabilise diluents
comprising biological molecules. In one embodiment, low molecular weight PLC
(less than 6000 Mw, preferably 640-5000) is used (for instance PLC with on
average
17 residues with a Mw of 2178). PLC is a fully bio-degradable polyamino acid
with a
pendent free y-carboxyl group in each repeat unit (pKa 4.1) and is negatively
charged at a pH7, which renders this homopolymer water-soluble and gives it a
polyanionic structure. PLC may be made using conventional peptide synthesis
techniques. It is also available from Sigma-Aldrich, St. Louis, MO, USA, in a
relatively polydisperse form (e.g. 17mers with a polydispersity around 2.6),
or from
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Neosystem, Strasbourg, France in a relatively monodisperse form (e.g. 8, 16,
24 or
32mers with a polydispersity close to 1).
PLC as used in the present invention will usually be at a concentration
between 10-2000pg/m1, i.e. 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125,
150,
175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,
900, 950,
1000, 1100, 1200, 1300, 1400, 1500, 1750 or 2000pg/ml.
Oligonucleotide
The oligonucleotides for use in the present invention may be composed of
ribonucleic acid, deoxyribonucleic acid or any chemically modified nucleic
acid
known in the art. However, the oligonucleotides utilised in the present
invention are
typically deoxynucleotides. The oligonucleotides may contain any sequence of
purines or pyrimidines.
In one embodiment the oligonucleotide comprises a CpG. CpG is an
abbreviation for cytosine-guanosine dinucleotide motifs present in DNA.
Historically,
it was observed that the DNA fraction of BCC could exert an anti-tumour
effect. In
further studies, synthetic oligonucleotides derived from BCC gene sequences
were
shown to be capable of inducing immunostimulatory effects (both in vitro and
in
vivo). The authors of these studies concluded that certain palindromic
sequences,
including a central CG motif, carried this activity. The central role of the
CG motif in
immunostimulation was later elucidated in a publication by Krieg, Nature 374,
p546
1995. Detailed analysis has shown that the CG motif has to be in a certain
sequence context, and that such sequences are common in bacterial DNA but are
rare in vertebrate DNA. The immunostimulatory sequence is often: Purine,
Purine,
C, G, pyrimidine, pyrimidine; wherein the dinucleotide CG motif is not
methylated,
but other unmethylated CpG sequences are known to be immunostimulatory and
may be used in the present invention.
In certain combinations of the six nucleotides a palindromic sequence is
present. Several of these motifs, either as repeats of one motif or a
combination of
different motifs, can be present in the same oligonucleotide. The presence of
one or
more of these immunostimulatory sequence containing oligonucleotides can
activate
various immune subsets, including natural killer cells (which produce
interferon 7 and
have cytolytic activity) and macrophages (Wooldrige et al Vol 89 (no. 8),
1977).
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Although other unmethylated CpG containing sequences not having this consensus
sequence have now been shown to be immunomodulatory.
In one embodiment of the invention, the oligonucleotide contains two or more
dinucleotide CpG motifs separated by at least three, preferably at least six
or more
nucleotides.
The oligonucleotides of the present invention are typically
deoxynucleotides. In a preferred embodiment the internucleotide bond in the
oligonucleotide is phosphorodithioate, or more preferably a phosphorothioate
bond,
although phosphodiester and other internucleotide bonds are within the scope
of the
invention including oligonucleotides with mixed internucleotide linkages.
Examples of preferred oligonucleotides have the following sequences. The
sequences preferably contain phosphorothioate modified internucleotide
linkages.
TCC ATG ACG TTC CTG ACG TT (CpG 1826;) - SEQ ID NO:1
TCT CCC AGC GTG CGC CAT (CpG 1758) - SEQ ID NO:2
ACC GAT CAC GTC GCC GGT CAC GGC ACC ACG - SEQ ID NO:3
TCC TCC TTT TGT CGT TTT GTC GTT (CpG 2006/CpG7909) SEQ
ID NO:4
TCC ATG ACG TTC CTG ATG CT (CpG 1668) SEQ ID NO:5
TCC ACG TTT TCC GCG CGC GCC G (CpG 5456) SEQ ID NO:6
TCC TCC TTT TGT CGT (CpG 15mer): SEQ ID NO: 9
TCC TCC TTT TGT CGT TTT GTC GTT TCC TCC (CpG 30mer):
SEQ ID NO:10
Alternative CpG oligonucleotides may comprise the preferred sequences
above in that they have inconsequential deletions or additions thereto.
The CpG oligonucleotides utilised in the present invention may be
synthesized by any method known in the art (eg EP 468520). Conveniently, such
oligonucleotides may be synthesized utilising an automated synthesizer.
Oligonucleotides for use in the present invention are usually 2-500 bases in
length, i.e. 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125, 150, 175,
200, 250, 300,
350, 400, 450, or 500 bases. In one embodiment the oligonucleotides for use in
the
present invention are 10-50 bases in length, i.e. 10, 11, 12, 13, 14, 15, 16,
17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 bases in length.
Oligonucleotides as used in the present invention will usually be at a
concentration between 10-2000pg/ml, i.e. 2, 5, 10, 15, 20, 25, 30, 35, 40, 50,
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100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,
750, 800,
850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1750 or 2000pg/ml.
Diluent
The term diluent refers to a diluting agent. In the context of the present
invention the
diluent may refer to the diluent alone, or it may refer to the diluent
comprising one or
more solutes. These solutes can be any molecule, including, but not limited to
salts,
buffers, detergents, polymers, proteins and/or oligonucleotides. The diluent
will
usually be water, but may also be another suitable solvent.
Diluent A
As described above, as PRAME is over expressed in E.coli, where it forms
inclusion bodies, in order to solubilise PRAME it is necessary to expose the
inclusion
bodies to strongly solubilising conditions requiring anionic detergent and
urea. It is
also necessary to keep PRAME soluble during the purification process. Diluent
A
may refer to the diluent which is used to directly solubilise PRAME from the
cells in
which it is expressed or it may refer to any buffer used during the
purification of
PRAME. The term "Diluent A" can be used to refer to the diluent irrespective
of the
presence of the polyanionic compound. As referred to herein, diluent A is any
diluent
used in the presently disclosed process for the purification of PRAME.
In one embodiment, diluent A will usually comprise a detergent. In one
embodiment, the detergent will usually be at a concentration less than 0.1%
w/v. In a
further embodiment the detergent will be an anionic detergent. An anionic
detergent
is any detergent in which the lipophilic part of the molecule is an anion;
examples
include soaps and synthetic long-chain sulfates and sulfonates. In one
embodiment
the anionic detergent is sodium dodecyl sulphate (SDS), sodium docusate or
lauryl
sarcosyl.
In one embodiment, diluent A comprises one or more of Tris, NaH2PO4.2H20,
urea and lauryl sarcosyl.
Where present the Tris will be at a concentration between 1-200mM, i.e. 1,2,
3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,
45, 50, 60,
70, 80, 90, 100, 125, 150, 175 or 200mM.
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Where present the NaH2PO4.2H20 will be at a concentration between 1-
200mM, i.e. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30,
35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200mM.
Where present the Urea will be at a concentration between 0.5-9M, i.e. 0.5,
1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5
or 9.0M.
Where present the lauryl sarcosyl will be at a concentration between 0.1-10%
w/v,
i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9, or
10%w/v.
Diluent B
As described above, in order to use PRAME in a composition for injection in
patients, it must be transferred to a suitable diluent. Such a diluent will
usually be
substantially free of the detergents used in the solubilisation and
purification of
PRAME. In one embodiment diluent B will be substantially free of detergent.
The term "substantially free" means that there will be less than 0.1% w/v of
detergent, i.e. 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01% or less
w/v of
detergent. In a further embodiment the term "substantially free" means that
there will
be less than 0.01% w/v detergent. i.e. 0.009, 0.008, 0.007, 0.006, 0.005,
0.004,
0.003, 0.002, 0.001%, 0.0005% or less w/v of detergent.
In one embodiment, diluent B comprises one or more of Borate and sucrose.
In one embodiment, diluent B comprises Borate and sucrose.
Where present the borate will be at a concentration between 1-200mM, i.e.
1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,
35, 40, 45,
50, 60, 70, 80, 90, 100, 125, 150, 175, or 200mM.
Where present the sucrose will be at a concentration between 0.1-20% w/v,
i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20% w/v.
Diluent C
Once the PRAME has been exchanged from diluent A to B, it may be
necessary to formulate PRAME into a new diluent, diluent C. For example,
diluent C
may be used to store PRAME, may be to allow lyophilisation of PRAME, or may be
for direct use in a patient.
In order to formulate PRAME in to diluent C, PRAME containing diluent B may
undergo diluent exchange with diluent C using the processes described above.
Additional components may be added to the PRAME containing diluent B in order
to
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arrive at a new diluent, diluent C. In addition or alternatively, diluent B
may be diluted
to arrive at diluent C. All of these methods are contemplated by the
invention.
In one embodiment, diluent C comprises one or more of Tris, borate, sucrose,
poloaxmer and CpG. In one embodiment, diluent C comprises Tris, borate,
sucrose,
poloaxmer and CpG.
Where present the Tris will be at a concentration between 1-200mM, i.e. 1,2,
3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,
45, 50, 60,
70, 80, 90, 100, 125, 150, 175 or 200mM.
Where present the borate will be at a concentration between 1-200mM, i.e.
1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,
35, 40, 45,
50, 60, 70, 80, 90, 100, 125, 150, 175, or 200mM.
Where present the poloxamer will be at a concentration between 0.01-2% w/v,
i.e. 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12,
0.13, 0.14,
0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25,0.26, 0.27,
0.28, 0.29,
0.30, 0.35, 0.40, 0.45, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.25, 1.50, 1.75,
or 2% w/v.
In one embodiment the poloxamer is poloxamer 188.
Where present the sucrose will be at a concentration between 0.1-20% w/v,
i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20% w/v.
Where present, the CpG will be at a concentration between 10-2000pg/ml, i.e.
2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300,
350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300,
1400, 1500, 1750 or 2000pg/ml.
Diluent C may be at a pH in the range of 5-10, i.e. a pH of 5, 5.5, 6.0, 6.5,
7.0,
7.5, 8.0, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8
9.9 or 10.
Diluent Exchange
Diluent exchange refers to the transfer of protein from a first diluent to a
second diluent. The protein may itself be transferred, but it is more common
for the
diluent to be transferred. Examples of diluent exchange include, but are not
limited
to, dialysis, Diafiltration and size exclusion chromatography.
As described herein, the aim of the invention is to reduce the aggregation of
a
protein during a diluent exchange. The methods of the invention refer to
adding a
polyanionic compound to diluent A prior to diluent exchange with diluent B.
The
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skilled person will appreciate, however, that there are situations where the
polyanionic compound can be added to diluent A contemporaneously with the
diluent
exchange. For example, the polyanionic compound may be present in diluent B.
Upon commencement of the diluent exchange, polyanionic compound present in
diluent B will be added to diluent A. In another example, the polyanionic
compound
may be added to a combination of diluent A and B after the diluent exchange
has
begun. Such situations are also contemplated by the invention.
Dialysis
Dialysis relies on the separation of particles in a liquid on the basis of
differences in their ability to pass through a membrane. For example, a small
volume
of diluent A containing a protein is placed into a semi-permeable membrane
which is
sealed. The membrane is then placed into a larger volume of a diluent B. The
membrane allows the movement of small solute molecules and solvent across the
semi-permeable membrane, but not the larger protein molecules. After a period
of
time, the diluent on the outside and inside of the membrane equilibrates.
Because of
the large difference in volume of the two diluents, equilibration effectively
results in
the replacement of diluent A with diluent B.
Dia filtration
Diafiltration is also a membrane based separation that is used to exchange
diluents. In batch diafiltration, diluent A is typically diluted by a factor
of two using
new diluent, i.e. diluent B, brought back to the original volume by tangential
flow
filtration (TFF), permeate elimination is used to reduce the volume to initial
value,
and the whole process repeated several times to achieve the elimination of
original
diluent A. In continuous diafiltration the diluent B is added at the same rate
as the
permeate flow.
Compositions
As described above, the problem identified and solved by the inventors of the
present application is related to the aggregation of PRAME. Transfer of PRAME
from
a diluent comprising a strong detergent to one which is substantially free of
detergent
causes the aggregation of PRAME. This aggregation continues over time and
eventually causes precipitation of the PRAME out of solution.
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The methods of the invention described above solve this problem and allow
the production of PRAME composition which has a consistent hydrodynamic
radius.
Accordingly, the invention provides a composition comprising PRAME and an
oligonucleotide, wherein PRAME has a particle size of 10-40nm, i.e. 10, 11,
12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35,
36, 37, 38, 39 or 40nm. In a further embodiment, PRAME has a particle size of
15-25
nm, i.e. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25nm. In a further
embodiment,
PRAME has a particle size of 16-20nm, i.e. 16.1, 16.2, 16.3, 16.4, 16.5, 16.6,
16.7,
16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0,
18.1, 18.2,
18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9,19.0, 19.1, 19.2, 19.3, 19.4, 19.5,
19.6, 19.7,
19.8, 19.9, or 20.0nm.
The invention also provides a composition comprising PRAME and an
oligonucleotide, wherein PRAME has a particle size as described above and a
polydispersity index between 0.1 and 0.4, i.e. 0.11, 0.12, 0.13, 0.14, 0.15,
0.16, 0.17,
0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30,
0.31, 0.32,
0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39 or 0.40nm. In a further embodiment,
PRAME
has a polydispersity index of between 0.2 and 0.3, i.e. 0.20, 0.21, 0.22,
0.23, 0.24,
0.25, 0.26, 0.27, 0.28, 0.29, or 0.30.
Both the hydrodynamic radius and the polydispersity can be measured by
dynamic light scattering.
Dynamic Light Scattering (DLS)
Dynamic light scattering (DLS), which is also known as photon correlation
spectroscopy (PCS) or quasi-elastic light scattering (QELS), uses scattered
light to
measure the rate of diffusion of protein particles in a solution. This motion
data is
processed to derive a size distribution for the sample, where the size is
given by the
"Stokes radius" or "hydrodynamic radius" of the protein particle. This
hydrodynamic
size depends on both mass and shape (conformation). Dynamic scattering allows
detection of the presence of very small amounts of aggregated protein (<0.01%
by
weight).
In dynamic light scattering the time dependence of the light scattered from a
very small region of solution, over a time range from tenths of a microsecond
to
milliseconds is measured. These fluctuations in the intensity of the scattered
light are
related to the rate of diffusion of molecules in and out of the region being
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(Brownian motion), and the data can be analyzed to directly give the diffusion
coefficients of the particles doing the scattering. When multiple species are
present,
a distribution of diffusion coefficients is seen.
Traditionally, rather than presenting the data in terms of diffusion
coefficients,
the data are processed to give the "size" of the particles (radius or
diameter). The
relation between diffusion and particle size is based on theoretical
relationships for
the Brownian motion of spherical particles, originally derived by Einstein.
The
"hydrodynamic diameter" or "Stokes radius", Rh, derived from this method is
the size
of a spherical particle that would have a diffusion coefficient equal to that
of the
protein.
Most proteins are not spherical, and their apparent hydrodynamic size
depends on their shape (conformation) as well as their molecular mass.
Further,
their diffusion is also affected by water molecules which are bound or
entrapped by
the protein. Therefore, the hydrodynamic radius can differ significantly from
the true
physical size (e.g. that seen by NMR or x-ray crystallography).
Hydrodynamic size and polydispersity index were determined by DLS. In one
embodiment, hydrodynamic size and polydispersity index were measured by
ZetaNano from Malvern.
Pharmaceutically acceptable compositions
The invention also provides a method of producing a pharmaceutically
acceptable PRAME solution comprising the steps of: (a) carrying out a diluent
exchange according to the methods of the invention; and (b) sterilising the
formulation produced in step (a).
In one embodiment, the method comprises an additional step (b') formulating
the protein into diluent C prior to step (b). In a further embodiment the
method
comprises the additional step (c) lyophilising the formulation produced in
step (b')
The sterilisation may be via any method known in the art including, but not
limited to,
UV sterilisation, heat sterilisation or filtration. In one embodiment the
sterilisation is
achieved using filtration. The filter will usually have a pore size of 0.05-
1.0pm, i.e.
0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0pm. In a further
embodiment, a
series of one of more filters may be used to achieve sterilisation and the
sterilisation
may occur at any point during the steps described above.
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Throughout this specification and the claims which follow, unless the context
requires otherwise, the word "comprise", and variations such as "comprises"
and
"comprising", will be understood to imply the inclusion of a stated integer or
step or
group of stated integers or steps but not to the exclusion of any other
integer or step
or group of integers or steps.
The invention will be further described by reference to the following, non-
limiting, figures and examples.
Examples
Example 1 ¨ Characterisation of PRAME
isoelectric point
The isoelectric point (IEP) of the PRAME antigen was determined on purified
antigen solubilised in 5mM Borate buffer pH 9.8 ¨ 3.15% sucrose by
electrophoretic
mobility measurement and Zeta potential calculation with ZetaNano@ from
Malvern.
The experimentally obtained value of 6.44 was very close to the value
calculated
from theoretical amino acid composition (6.41). As the pH of reconstituted
vaccine in
adjuvant system A (ASA) (Sorbitol) is 8.0, it is expected that the antigen,
PRAME,
and the CpG present will be globally negatively charged. Therefore, no
electrostatic
interaction was expected to occur between the two entities.
Material and method for IsoElectric Point (IEP) determination:
Samples were diluted in 5 mM Borate buffer pH 9.8 ¨ 3.15% sucrose, and the
pH was adjusted to the desired pH with HCI and/or NaOH. The reported zeta
potential is the average of 5 consecutive measurements. IEP is the pH at zero
zeta
potential in the measured "zeta potential versus pH" curve (Figure 1/21). A
reference
standard is tested to check performance of the equipment and measurement
cells.
The sample measurements were conducted using the experimental conditions
shown in Table 1:
Table 1:
Laser wavelength: 633nm
Laser power: 4mW
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Scattered light detected at: 13
Temperature: 22 C
Duration: automatic determination by the soft
Number of consecutive 3-5 consecutive measurements
measurements:
pH adjusting solutions if IEP HCI 0.3M, NaOH 0.5M
required
Aggregation state of PRAME
Aggregation profile
As part of the proposed plan for stability, the aggregation state of purified
PD1/3-PRAME (SEQ ID NO:8)-His bulk is monitored by:
= Dynamic Light Scattering (DLS) analysis and
= Size-Exclusion High-Performance Liquid Chromatography (SEC) coupled to
Multi-Angle Laser Light Scattering (MALLS) detection and to a concentration
sensitive detection such as a refractive index (RI).
Purified bulks (PB) of PD1/3-PRAME-His were also characterized by
Sedimentation Velocity profiling using Analytical Ultracentrifugation (SV-
AUC).
Size analysis by DLS
Hydrodynamic size and polydispersity index were determined by DLS for each
purified PD1/3-PRAME-His bulk at release (TO). For lots DPRAAPA003,
DPRAAPA004 and DPRAAPA005, the values of hydrodynamic size (Z-average, nm)
and polydispersity were reproducible between the batches. Antigen is
aggregated
with a size between 16.6 and 19.9 nm; polydispersity ranges from 0.218 to
0.284.
No significant change in size can be detected by DLS when the PB lots
DPRAAPA003, DPRAAPA004 and DPRAAPA005 are either incubated for 4 hours at
4 C or stored for 12 months at -70 C (see m3.2.S.7.3).
SEC-MALLS analysis
Method of analysis:
SEC analysis using UV, MALLS and RI detectors allows determination of the
absolute molar mass (MM) and size (hydrodynamic radius or Rh in nm) of
polymers
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or biopolymers in solution without reference to calibration standards and
without prior
assumptions about their molecular conformation. It is also a sensitive method
for
detecting aggregates even in a small amount.
Results and discussion:
This analysis was performed on the PB lots DPRAAPA003, DPRAAPA004
and DPRAAPA005. Figure 2/21, Figure 3/21 and Figure 4/21 show the light
scattering (LS) profiles, RI profiles, and molar mass (MM) distribution in
function of
the elution volume obtained by SEC-MALLS analyses of the three GMP lots at
release.
In the final buffer (5 mM borate, 3.15% sucrose, pH 9.8), PBs consist of
polydisperse, soluble aggregates eluting between 6.0 and 7.7 mL and by MM
values
varying between 600 and 3,000 kDa.
Aggregates with higher molecular mass elute between 5.0 and 6.0 mL, but
they represent a small fraction of the total purified protein bulk. This was
confirmed
by SV-AUC analysis shown below in Figure 5/21 and Table 2.
SV-AUC analysis
Method of analysis:
To ensure that the aggregation profile of a biopolymer in solution is not
influenced and/or caused by putative interactions between the solution to be
analyzed and the chromatographic bed, the protein aggregation status and
distribution was analyzed directly in solution and in real time by analytical
ultracentrifugation. Briefly, the reference (protein buffer) and sample
solutions are
centrifuged at high speed (35,000 rpm) and their absorbance at 280 nm
recorded.
The acquired data reflect the spatial concentration gradients of sedimenting
species
and their evolution with time generated after applying the centrifugal field.
Sedimentation depends both on the size and shape of the protein. Time course
analysis of the sedimentation process also termed sedimentation velocity (SV-
AUC)
allows the calculation of the sedimentation coefficients (s). The s values are
reported
in Svedberg (S) units, one unit corresponding to 10-13 seconds.
For purified PD1/3-PRAME-His bulk, the sedimentation coefficient distribution
c(s) were obtained using Sedfit software.
Results and discussion
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Figure 5/21 shows the results of the SV-AUC analysis performed on PB lots
DPRAAPA003, DPRAAPA004 and DPRAAPA005 at release.
Table 4 shows the correspondence between each aggregate detected in Figure
5/21
and its respective sedimentation coefficient and molecular weight. This
qualitative
interpretation is based on the fact that the 72-kDa monomeric PD1/3-PRAME-His
protein forms globular compact aggregates as demonstrated by electron
microscopy.
For such globular aggregates, a classical frictional ratio f/f of 1.2 can be
attributed.
Table 2: Correspondence between the aggregates and their
sedimentation coefficient and molecular weight.
Aggregate 1 2 3 4 5 6 8 10 12 2r,
_lc
Number IN)
Sedimentation
42 - '2- 17 2.-1 22 :I
51:
coeff cient IS)
IMQ. *Da! 72 576 72C -%4 -
As shown in Figure 5/21 and in Table 2, the majority of purified protein bulk
is
represented in solution by a polydisperse population (characterized by
sedimentation
coefficients between 3.6 and 30 S) ranging from the 72-kDa monomeric form to
aggregated complexes consisting of 20 monomeric molecules (MW = 1,440 kDa).
The mean sedimentation coefficient (s bar) obtained at release for PB from
lots
DPRAAPA003, DPRAAPA004 and DPRAAPA005 were 13.5, 10.2 and 11.1 S,
respectively.
The 3.6 - 30-S polydisperse population accounts for 95%, 97% and 96% of
the total PB from lots DPRAAPA003, DPRAAPA004 and DPRAAPA005,
respectively. The remainder is represented by higher aggregates characterized
by
higher sedimentation constants (30 to 60 S).
In conclusion, the three GMP lots of PD1/3-PRAME-His have similar
sedimentation coefficient distributions.
Example 2 ¨ Evidence of interaction with CpG
SDS-Page/WB anti-PRAME (additional band 7kDa above monomer)
SDS-PAGE analysis was conducted on Final Container reconstituted in ASA
(Sorbitol). As illustrated in Figure 6/21, additional band (band 1) is
detected in Final
Container at TO (cf. lane 3). Based on analysis by densitometry (Biorad GS-700
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Imaging densitometerTm), this additional band is characterized by a MW of 7
kDa
higher than PRAME monomer band (band 2) and its intensity increases over time
but remains below 4% (w/w versus monomer) 96h post-reconstitution. Western
Blot
analysis using specific anti-PRAME antibody (Figure 7/21) confirmed that the
additional band is product-related and that its intensity slightly increases
over time
(lane 3 vs. 7).
Isothermal titration calorimetry
ITC measures directly the energy (heat) associated with a chemical reaction
triggered by the mixing of two components. A typical ITC experiment is carried
out by
the stepwise injection of a solution containing one reactant into the reaction
cell
containing the other reactant. The ITC setup used for the study of the PRAME
antigen / CpG complex implied the injection of CpG liquid bulk (diluted in the
reconstituted vaccine buffer (borate 5 mM sucrose 3.15% pH 9.8)) into a
solution of
PRAME antigen (in the same buffer). A typical titration profile is presented
in Figure
8/21.
As observed in Figure 8/21, each injection of CpG into the PRAME solution
results in a negative peak indicating a very significant exothermic binding
reaction.
Because the amount of uncomplexed protein available progressively decreases
after
each successive injection, the magnitude of the peaks becomes smaller until
complete saturation is reached. Of note, control experiments (data not shown)
consisting in the injection of PRAME buffer without the antigen gives a flat
profile.
The amount of CpG needed to reach the plateau of saturation is equivalent to
a mass ratio CpG/ antigen ranging between 0.05 and 0.10 in good agreement with
the complex stoichiometry determined by ultracentrifugation.
Materials and Methods
The isothermal titration calorimeter is composed of two identical cells made
of
a highly efficient thermal conducting material. Temperature differences are
monitored between a reference cell (filled with water) and a sample cell
(containing
the oil-in-water emulsion, AS03). Measurements consisted of time-dependent
input
of power (expressed as pcal/s) required to maintain equal temperatures between
the
reference and sample cells. Set up and general protocol used for the ITC
instrument
follow specifications provided by the manufacturer (MicroCal, USA). All
samples prior
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to use were degassed for 5 minutes to minimise data interference due to the
presence of bubbles. CpG was filled into the injection syringe and titrated
into the
sample cell containing the antigen. Titration comprised of 1 injection of 2p1
followed
by 24 successive injections of 10 pl, with a 6 minute delay between each
injection.
The antigen was loaded into the sample cell up to the fill level (the sample
cell in this
instrument has an internal volume at the fill level of 1404p1).
Control titrations for CpG and antigen alone are always included in the
testing
protocols.
Rate-zonal ultracentrifugation
In the aim of isolating a PRAME/CpG complex from free antigen and CpG,
rate zonal configuration was performed. Samples were loaded on top of a linear
sucrose gradient and separated based on their sedimentation rate. Unlike ITC
as
described above, this setup additionally allows the analysis of reconstituted
vaccine
samples. After optimization of the experimental conditions, distribution of
antigen and
CpG in a sucrose gradient was observed as shown in Figure 9/21.
Subsequently, SDS-PAGE was replaced by RP-HPLC-UV to obtain a
quantitative determination of the antigen in the sucrose fractions. To
determine
whether the CpG / antigen interaction is subject to significant batch-to-batch
variations, three repro lots containing 500 pg of PRAME per dose and 3 lots of
100
pg/dose were submitted to rate zonal ultracentrifugation and further analyzed
to
determine the stoichiometry of the CpG/antigen complex. These are shown in
Figure
10/21.
The results show that the amount of CpG associated with the antigen is very
similar between lots. As expected, decreasing the antigen dose from 500 to 100
pg
leads to a decrease of the amount of CpG in the complex. The stoichiometry, as
expressed by the CpG/Ag mass ratio is however not directly proportional to the
antigen dosage. The pre-incubation of the sample at 25 C for 24h doesn't
influence
the complex stoichiometry.
These results strongly suggest that CpG consistently interacts with PRAME.
Materials and methods
In to a 14 x 89 mm centrifuge tube, 5m1 of a 25 % sucrose solution was added
under the same volume 5m1 of a 5% sucrose solution. The tube was loaded on the
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Master Gradient to proceed a continuous gradient 5-25%. A volume of 1m1 was
removed on the tube of the gradient. This volume was replaced at the bottom of
the
gradient by 1 ml of 50% sucrose solutions. Samples (or controls) were pre-
incubated for 15 min at 25 C to allow all samples, including those stored at
4 C, to
begin the centrifugation at equivalent temperatures. 250p1 ml aliquot of the
sample
was loaded on the top of the gradient. The gradient was centrifuged at 100 000
relative centrifugal force (rcf) for 67 h at 4 C.
Sucrose Fraction collection
Fractions resulting from ultracentrifugation were collected by pipetting from
the top of the tube. Successive suction of 1-mL fractions was performed. Upon
collection, the fractions were stored at 4 C until subsequent analysis
Fraction analysis
Detection of ASCI antigens
Antigens were analyzed by SDS-PAGE. Alternatively, RP-HPLC-UV was
employed for quantitative purpose.
Detection of liposome components.
Liposomes localization was performed by determination of cholesterol
(colorimetric kit, Roche Diagnostics). Alternatively, IP-HPLC-UV was used.
Detection of CpG
CpG was determined by IEX-HPLC-UV.
Samples
Antigen PBs: Prame: R03.
Final containers (lyophilized cakes): Prame: 08H14PRA01, 08H2OPRA01,
08109PRA01 (500pg/HD)
08H14PRA02, 08H2OPRA02, 08109PRA02 (100pg/HD). ASO1B:DA1BA008A
CpG liquid bulk: DCPGAFA003
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ELISA (interference) - PD-PRAME-his : antigen content by ELISA on Ph I/11
material
In order to further investigate the observed interaction between PRAME and
CpG a sandwich ELISA based on the use of a mAb anti-PRAME and a polyclonal
antibody (Pab) anti protein D (PD) was developed to measure the antigen
content.
Using this ELISA to test the antigen content of the PB gives the expected
value of PBs (PB). However, when the ELISA is applied to Final Container (FC),
a
loss of antigenicity is seen.
N.B. The PB contains the antigen in a Borate 5mM sucrose 3.15% buffer while
the
FC contains CpG (420pg/dose), poloxamer 188 at 0.24%, sucrose 4% and Tris
16mM
To test whether this observed effect was related to the CpG, the PB was
spiked with increasing doses of CpG and the antigen content was measured by
ELISA, as shown in table 3.
Table 3:
expected conc. result recovery
non lineanse (Lowry)pg/m1 ELISA (pg/m1)
NELISA/LowrY)
A std RO1 1604pg/nnl 1604 1594
99
B R02 1851pg/m1 + CpG 1000pg/m1 1851 67
4
C R02 1851pg/m1 + CpG 100pg/m1 1851 489
26
D R02 1851pg/nnl + CpG 10pg/m1
1851 1427 77
E R02 1851pg/nnl + CpG 1pg/m1 1851 1697
92
F R02 1851pg/nnl + CpG 0.1pg/m1 1851 1448
78
G R02 1851pg/m1 + CpG 0.01pg/m1
1851 1542 83
H R02 1851pg/m1 1851
1485 80
These results show that the addition of CpG in the PB is associated with
decreases the antigenicity especially from the concentration comprised between
10
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and 100pg/m1 suggesting a change of conformation of the antigen when CpG is
added.
Material and method
This method is based on a "Sandwich" ELISA:Before addition of the antigen
PDPRAME-his (repro lot R02) the immunoplate is coated with a mouse monoclonal
antibody directed against PRAME (MK1H8C8 diluted 500x) overnight at 4 c. After
reaction with the antigen for 90' at 37 c, a rabbit polyclonal antibody
directed against
PD (LA598733) is added for 90' at 37 c. After reaction with the Pab for 90' at
37 c, a
biotinylated donkey whole antibody against rabbit immunoglobulins is added for
90'
at 37 c. The antigen-antibody complex is revealed by incubation with a
streptavidin-
biotinylated peroxidase complex for 30' at 37 c. This complex is then revealed
by the
addition of tetramethyl benzidine (TMB) for 15' at Room Temperature and the
reaction is stopped with 0.2 M H2504. Optical densities are recorded at 450
nm.
The concentrations of samples are calculated by SoftMaxProTm referring to a
standard antigen (repro lot RO1 at 1604pg/m1)
SEC-HPLC (interference)
Material and Methods
Size-exclusion chromatography (SEC) also called gel permeation or gel
filtration chromatography is a method separating molecules in solution based
on their
size or shape. Antigen size follow-up through formulation process is one of
the
success criteria when developing a vaccine candidate. The first objective was
therefore to develop an analytical SEC method for this purpose. As CpG is
added to
the vaccine candidate, purified antigen alone (Figure 11/21) or spiked with
CpG
solution (Figure 12/21) was injected on several SEC columns (alone or in
combination) from Tosoh supplier equilibrated in 5 mM Borate buffer pH 9.8 ¨
3.15%
sucrose (= buffer of the purified antigen) at a flow-rate of 0.5 ml/min and
with UV
detection at 220 nm. Guard column recommended by Tosoh supplier was also used
with each column or combination of columns. As illustrated in Figure 12/21,
injection
of purified antigen spiked with CpG solution on a single column (TSK G5000
PWx1 or
TSK G6000Pwx1) led to significant overlapping of molecules peaks while a
combination in series of two columns improves the resolution. Combination of
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columns, TSK G4000PWx1 + G 6000 PWx1 was therefore selected as SEC analytical
tool for the follow-up of formulation development.
Characteristics of the columns tested
= TSKgel
PWxITM guard column: 6 mm internal diameter (ID) x 4 cm length (L) ¨
Tosoh - Ref 08033
= TSK G4000 PWxITM column: 7.8 mm ID x 30 cm L ¨ Tosoh - Ref 08022
= TSK G6000 PWxITM column: 7.8 mm ID x 30 cm L ¨ Tosoh - Ref 08024
= TSK G5000 PWxITM column: 7.8 mm ID x 30 cm L ¨ Tosoh - Ref 08023
As illustrated in Figure 13/21, increase of retention time and surface area
for
purified antigen (1.68 mg of protein/m1) is already observed after spiking
with CpG
solution up to 10 pg/ml of. The higher retention time would suggest a lower
size
(=indirect indication of the positive effect of CpG on antigen solubility).
Purified
antigen was therefore further spiked with increasing concentrations of
oligonucleotide solution (from 10 up to 1050 pg/ml). Surface area of first
elution peak
(peak 1) increases for CpG concentrations up to 60 pg/ml, then remains
constant for
all upper spiking concentrations up to 1050 pg CpG/ml. A second peak (peak 2)
corresponding to free CpG (Figure 14/21 representing UV profiles obtained
after
injection of different concentrations of CpG solution) is detected from 180
pg/ml of
CpG.
Example 3 ¨ Excipient screening
Starting from purified antigen solubilised in 5 mM Borate pH 9.8 ¨ sucrose
3.15%, 25 excipients were assessed for the stabilization of the antigen size
upon
storage at +4 C and at +22 C. The listing of the excipients and the
concentrations
tested are listed in the table 4
First selection of the candidates was performed through visual observation
and size analysis by dynamic light scattering after 24h storage at 22 C (cf.
table 4
and Figure 15/21). Amongst all the excipients tested, only four of them
allowed
antigen size stabilization: SDS 0.01%, Sodium Docusate 0.01%, Sarcosyl 0.03%
and
CpG (from 20pg/m1 to 50pg/m1)
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Table 4: Listing and concentrations of the excipients tested for the
stabilization of the
antigen size and visual observation on purified antigen samples spiked or not
with
excipient after 24h storage at 22 C. PB = Purified antigen in 5 mM Borate pH
9.8 ¨
sucrose 3.15%.
Table 4:
Excipient Concentration unit Visual
observation
24h22 C
L-glycine 10 mM cloudy
100 mM opaque
L-Histidine 10 mM cloudy
100 mM opaque
L-Glutamic acid 1 mM slightly cloudy
mM cloudy
L-Aspartic acid 0.5 mM slightly cloudy
100 mM opaque
L-Leucine.2HCI 5 mM slightly cloudy
30 mM cloudy
MgC12.6H20 1 mM opaque
50 mM opaque
MgSO4.7H20 1 mM opaque
50 mM opaque
Trehalose.2H20 1 cyo slightly cloudy
5 cyo cloudy
Polyethyleneglycol 300 1 cyo slightly cloudy
5 cyo cloudy
Polyethyleneglycol 6000 1 cyo cloudy
5 cyo cloudy
(Myo-)Inositol 1 cyo cloudy
5 cyo very cloudy
D-Mannitol 1 cyo opaque
5 cyo opaque
Sorbitol 1 cyo opaque
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5 cyo very cloudy
SDS Sodium Lauryl Sulfate 0.01 cyo clear
0.03 cyo clear
Sodium docusate 0.01 cyo clear
0.03 cyo clear
Solutol H515 0.01 cyo very slightly
cloudy
0.05 cyo very slightly
cloudy
Triton X-100 (Octoxynol 9) 0.3 cyo slightly cloudy
0.5 cyo slightly cloudy
Poloxamer 188 (Lutrol F68) 0.05 cyo slightly cloudy
0.5 cyo slightly cloudy
SulfobetaIne (5B3-12) 0.01 cyo slightly cloudy
0.03 cyo slightly cloudy
2-Pyrrolidone 0.01 cyo slightly cloudy
0.05 cyo slightly cloudy
Propyleneglycol 0.1 cyo slightly cloudy
1.5 cyo slightly cloudy
a-tocopheryl 1 mM clear
Hydrosuccinate (Vit E
succinate)
5 mM clear
CPG 0 pg/m1 slightly cloudy
20 pg/m1 slightly cloudy
30 pg/m1 slightly cloudy
35 pg/m1 slightly cloudy
40 pg/m1 slightly cloudy
45 pg/m1 slightly cloudy
50 pg/m1 slightly cloudy
Sarcosyl 0.03 cyo slightly cloudy
Purified antigen (= PB) 1500 pg/m1 slightly cloudy
1250 pg/m1 slightly cloudy
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I I 1125
1 pg/ml slightly cloudy I
Additional stability data were generated on the four selected candidates (SDS,
Sodium Docusate, Sarcosyl and CpG) up to 14 days at 4 C. As illustrated by the
Figure 16/21, antigen size remains stable in presence of the four excipients
while it
increases up to 84 nm for the non-spiked purified antigen (= PB sample).
Turbidity
measurement on the same samples (cf. Figure 17/21) confirmed an evolution only
for the non-spiked purified antigen.
Next step included the evaluation of ASA (Sorbitol) compatibility (Liposome
size and Q521 quenching) with the ionic detergents (Sarsosyl, SDS and Sodium
Docusate). Liposome size increases in presence of 1% SDS or Sodium Docusate
and remains stable up to 1% of Sarcosyl (cf. Figure 18/21). The three ionic
detergents alone induce lysis of red blood cells.
Conclusions:
Taking into account that SDS, Sodium Docusate and Sarcosyl alone induced
red blood cells lysis and the limited or non injectability of these
excipients, the use of
CpG as an excipient in the PB was prioritized.
Example 4¨ Effect of CpG on PRAME PB solubility
Introduction
This example summarizes the data collected to document the solubilizing
effect of the CpG7909 on PRAME antigen in final purification buffer.
When put in the final buffer (5mM Borate ¨ 3.15% Sucrose buffer) it was
observed that PRAME antigen was very likely to form insoluble aggregates and
cause precipitation. An effort was made to find an excipient to enhance
solubility of
the protein. Among a panel of excipient candidates, CpG was evaluated and
demonstrated an (unexpected) improvement of PRAME solubility in the final
buffer.
The results of CpG concentration screening experiment are described below.
First CpG concentration screening ¨ Dialysis trials
Experimental design
The aim is to start with a sample of PB of PRAME in a Borate-Sucrose buffer
containing 300ppm Lauryl-Sarcosyl (LS). This amount of detergent has
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demonstrated its ability to keep the protein soluble. Then we use a dialysis
operation
to eliminate the LS and replace it with an increasing amount of CpG. After the
buffer
exchange, the aggregation evolution of the product is monitored by DLS to
estimate
the amount of CpG needed to maintain a steady aggregation state.
Material & Buffers
Starting Material: PB PRAME in buffer 5mM Borate / 3.15% sucrose / 300ppm
Lauryl-Sarcosyl ¨ pH 9.8 (designated R23/1). Four samples of 2m1 of PB were
spiked up to tested concentration using a concentrated solution of CpG: 0
pg/ml CpG
(control); 50 pg/ml CpG; 200 pg/ml CpG; 400 pg/ml CpG. Dialysis buffer = 5mM
Borate /3.15% Sucrose ¨ pH 9.8 (2 x 1L per assay). Dialysis cassette [Pierce
Slide-
A-Lyzer 20,000 MWCO]
Method
2 ml of sample were introduced in a dialysis cassette. Each cassette was
submerged in a recipient containing 1L of dialysis buffer. The dispositive was
put
under gentle agitation (magnetic stirrer) at room temperature.
The first 1L of dialysis bath was replaced by 1L of new buffer after 2 hours
and left under gentle agitation overnight at room temperature.
The following day, the sample in the cassette was recovered in an eppendorf
container (PP) and stored at +4 C for further analysis.
Analvtics
Analysis was performed by: visual aspect; CpG content by HPLC-IEX-UV
(Dionex DNAPac PA200TM column) to monitor the remaining CpG content; Lauryl-
Sarcosyl content by RP-HPLC-UV (Waters SunFire C18 column) to insure that LS
(initial solubilisating detergent) is well removed; Dynamic Light Scattering
(DLS)
(ZetaNano@ from Malvern) is measured on dialysed product after a 24h and a 72h
storage period at +4 C to follow-up size evolution.
Results
Visual aspect: all samples are limpid after dialysis operation
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Table 5: Size by DLS after 24h and 72h / +4 C
After 24h/RT dialyse After 72h/+4 C
Polydispersit Z-
Sample Z-ave(nm) y index (Pd I) ave(nm) Pdl
Negative Ctrl
(no dialyse) 22.8 0.17
+ 0 pg CpG
(Positive ctrl) 56.6 0.134
0.1
+ 50 pg CpG 24.9 0.18 27.6 7
+ 200 pg 0.2
CpG 20.4 0.21 20.6 6
+ 400 pg 0.2
CpG 20.4 0.21 21.04 4
Table 6: Lauryl-Sarcosyl content by RP-HPLC + CpG content by IEX-HPLC
LS CpG CpG
content content Recovery
Sample ppm (pg/ml) %
PRAME Control (TO - no
dialysis) 293
PRAME + 50 pg CpG
(+dialysis) <0.5 39 78
PRAME + 200 pg CpG
(+dialysis) <0.5 153 77
PRAME + 400 pg CpG
(+dialysis) <0.5 317 79
B03-Sucrose + 100 pg CpG
(no dialysis) 94.2 94
B03-Sucrose + 100 pg CpG
(+dialysis) 71.6 72
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The LS is well eliminated during dialysis operation (measure below LOQ for
dialysed
sample) and the CpG stays on the inner side of the dialysis cassette with an
average
recovery around 80% even in absence of PRAME.
We observed that a quantity of CpG between 50 and 200 pg/ml maintains the
particle size at approximately 20nm after removal of Lauryl-Sarcosyl. This
observation suggests that the PRAME-CpG interaction is beneficial to the
solubility
of PRAME.
Second CpG concentration screening ¨ UltraFiltration trials
Objective
The screening of the CpG quantity required to keep PRAME soluble using an
UltraFiltration system for assessment.
Material & Buffers
Starting Material: HydroxyApatite Flow-through (HA-FT) Antigen fraction from
R25/2
purification.
Sample Buffer composition = 20mM Tris - 6M Urea ¨ 0.5% Lauryl Sarcosyl ¨ 50mM
PO4 - ¨80mM Imidazole
4 samples of approximately 70 ml of HA-FT are processed in 4 independent UF
experiments. A small volume of aqueous concentrated solution of CpG is added
to
reach following concentration: 50 pg/ml CpG
UF-A (no CpG in Diafiltration buffer);
75 pg/ml CpG UF-B (no CpG in Diafiltration buffer); 100 pg/ml CpG
UF-C (no
CpG in Diafiltration buffer); 50 pg/ml CpG
UF-D (50pg/m1 CpG in Diafiltration
buffer)
The CpG spiked samples are incubated 1h at Room Temperature under very mild
agitation prior Ultrafiltration
Diafiltration buffers : 5mM Borate / 3.15% Sucrose ¨ pH 9.8 (for UF-A/B/C);
5mM
Borate /3.15% Sucrose + 50pg/m1 CpG - pH 9.8 (for UF-D)
UltraFiltration cassette [MinimateTm ¨ Omega ¨ from Pall Cut-off 30kD] ¨
surface
50cm2
UltraFiltration system [KrossFloTM from JM JM Bioconenct II]: including a
peristaltic
pump, appropriate tubing to accommodate the UF cassette and 3 pressure gauges
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Method
70m1 of HA-FT sample spiked to appropriate CpG content are diafiltered against
15
Diafiltration Volume (Vol total Diaf buffer = 1050m1) of diafiltration buffer.
UF-conditions:
Recirculation flow-rate = 35m1/min
TMP regulation : 8psi for DV 1 ¨> 8 then 12 psi for DV 9 -> 15 by adjusting
retentate
counter-pressure valve
No concentration of Ag is done - only diafiltration
At end of operation: final retentate product is kept at +4 C for further
analysis
A clean in place (CIP) of 2 x 30 min under NaOH 0.5N (static) is done between
different UF
Analytics
Analysis was performed by: CpG content by HPLC-IEX-UV (Dionex DNAPac PA200
column); Lauryl-Sarcosyl content by RP-HPLC-UV (Waters SunFire C18 column);
Dynamic Light Scattering (DLS) (ZetaNano from Malvern) is measured directly
after UF and after 1 week stored at +4 C and Room Temp to follow-up size
evolution.
Results
Table 7: DLS measures for each of the concentrations tested ¨ "$" represent s
the sample which comes from the UF run with the corresponding letter as
described above.
Z-
average
Buffer Conditions Stability Point (nm) Pdl
$A + 50pg/m1 CpG To 22.47 0.102
$A + 50pg/m1 CpG 1w/RT 23.85 0.095
$A + 50pg/m1 CpG 1w/+4 C 24.19 0.106
$B + 75pg/m1 CpG To 18.11 0.121
$B + 75pg/m1 CpG 1w/RT 18.98 0.104
$B + 75pg/m1 CpG 1w/+4 C 26.55 0.289
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SC + 100pg/m1CpG To 16.62 0.135
$C + 100pg/m1CpG 1w/RT 16.29 0.108
$C + 100pg/m1CpG 1w/+4 C 16.02 0.134
$D + Tp Diaf : 50
pg/ml CpG To 10.61 0.247
$D + Tp Diaf : 50
pg/ml CpG 1w/RT 11.28 0.221
$D + Tp Diaf : 50
pg/ml CpG 1w/+4 C 10.84 0.289
Table 8: LS content & CpG content
LS CpG
content content Recovery
Sample ppm (pg/ml) 0/0
Internal Control (150
ppm buffer) 161 - -
$A + 50 pg CpG <0.5 40 80
$B + 75 pg CpG- 49 65
$C + 100 pg CpG- 79 79
LS is fully removed after 15 diafiltration volume (even in presence of CpG).
CpG
recoveries measured between 65 ¨ 80%
Conclusions
The solubilizing effect of CpG was observed when using an Ulrafiltration.
The green arrow in Figure 19/21 represents a spiking concentration of
100pg/m1 CpG in HA-FT before UF-R is selected as the appropriate concentration
because the samples are the most stable overtime, i.e. there is no increases
in size
after 1 week.
It does not appear to be necessary to add CpG in diafiltration buffer (UF-D)
as
the majority of CpG remains on sample side and is sufficient to keep product
soluble.
Spiking of HA-FT with 100pg CpG on a 1L scale purification was then
investigated.
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Verification of 100pg CpG spiking option at 11_ scale
Objective
Verify the feasibility of a spiking with 100pg/m1 CpG with a 1L scale process
(final development scale)
Procedure
= Run R26/1 : full PRAME purification at 1L scale + spiking with 100pd/ml
CpG
in HA-FT prior UF
= Check stability of final product with classical stress tests
= Stability 1week @ -70 C! +4 C! RT and 37 C
= 2 to 3 Freeze / Thaw cycles (-70 C ¨ RT)
= check LS removal by RP-HPLC-UV
= check CpG content (IEX-HPLC-UV) at end of UF in 1L scale condition
Results
Table 9: Size evolution follow-up by DLS measures
Stress
Conditions Zav (nm) Pdl
TO 18.7 0.14
1w / -70 C 19.6 0.15
1w / -F4 C 21 0.24
1w / RT 20.9 0.2
1w / 37 C 19.2 0.1
2 cycles F/T 19.6 0.17
3 cycles F/T 40.9 0.29
There was no significant increase in the particle size (Zav = 18.7 ¨ 20.9nm)
after
1w/+4-RT-37 C and 2 F/T cycles. The increase in particle size after 3 F/T
cycles is
more significant. CpG content = 101 pg/ml. LS content < 0.5pg/m1
Conclusions
The R26/1run indicates that a 100pg/m1 CpG spiking in HA-FT is appropriate for
at
the 1L scale and addresses the precipitation of PRAME. (Figure 20/21)
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Example 5 - Effect of PLG and additional CpG oligonucleotides on PRAME PB
solubility
CPG and PLG solutions preparation and quantification
Stock solutions of CpG 15-mers and 30-mers were prepared at 30 mg/ml in water
(Stock solution of CpG-24mers already available at 30 mg/ml). Stock solution
of
PolyGlutamate (PLG)-24mers was prepared at 10 mg/ml in water. The three stock
solutions were filtered on 0.22 pm PVDF membranes (millex CV). CpG content in
the stock solutions was determined by RMN analysis. The content is 30.20mg/m1
for
CpG 15-mers and 29.34mg/m1 for CpG 30-mers. PolyGlutamate (PLG)-24mers
content was based on weighing.
Dialysis trials
A Dialysis step is proposed to remove the original solubilizing agent (Lauryl
Sarcosyl
¨ required to maintain PRAME solubility) and replace it by an alternative
candidate
under evaluation.
Experimental Conditions:
Starting sample : 2m1 of PRAME Purified Bulk in 5mM Borate ¨ 3.15% sucrose ¨
300
ppm Lauryl Sarcosyl ¨ pH 9.8 buffer.
According to experimental plan, some samples are spiked with candidate
solubilizing
agent (see Table 10)
- Dialysis membrane cut-off: 20 kDa
- Dialysis buffer: 2 x 1L of 5mM borate ¨ 3.15% sucrose ¨ pH 9.8.
According to experimental plan, some buffers are spiked with candidate
solubilizing agent (see Table 10).
- Each sample is dialysed against 1L buffer under gentle agitation for 2h
at room
temperature. Then the buffer is renewed and dialyse is pursued under gentle
agitation overnight at room temperature.
- Volumes of 2.0m1 to 2.1m1 for each sample were recovered after dialyse
(negligible dilution effect).
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Table 10: Dialysis trials
Sample Sample spiking Dialysis buffer
Sample
Reference
No spiking
No dialysis (sample stored at - 70 C) A
,
No spiking No dialysis (sample left
overnight at RT)
No spiking 5 mM Borate buffer pH 9.8 ¨
a,
0.
3.15% sucrose
a
Spiking with CpG15-mers
mM Borate buffer pH 9.8 ¨
solution up to 100 pg/ml
3.15% sucrose ¨ 100 pg/ml
CpG 15-mers
U) Spiking with CpG24-mers 5 mM Borate buffer pH 9.8 ¨
solution up to 100 pg/ml 3.15% sucrose ¨ 100 pg/ml
CpG 24-mers
5
co Spiking with CpG30-mers mM Borate buffer pH 9.8 ¨
solution up to 100 pg/ml 3.15% sucrose ¨ 100 pg/ml
CpG 30-mers
4c,
Spiking with PLG-24mers 5 mM Borate buffer pH 9.8 ¨
solution up to 100 pg/ml 3.15% sucrose ¨ 100 pg/ml
PLG 24-mers
(c)
Spiking with CpG15-mers
solution up to 100 pg/ml
'k Spiking with CpG24-mers
k solution up to 100 pg/ml 5 mM Borate buffer pH 9.8
3.15% sucrose
Spiking with CpG30-mers
solution up to 100 pg/ml
Spiking with PLG-24mers
(, solution up to 100 pg/ml
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Analysis
PRAME content by RPC
PD1/3-Prame-His content is determined using a Reverse-Phase High Performance
Liquid Chromatography system coupled with a UV detector. Standards and samples
are diluted in the appropriate buffer prior to pre-treatment in Sodium Dodecyl
Sulfate
solution.
Detection of PD1/3-Prame-His is performed at 214 nm. Calibration curve is
prepared
with a PD1/3-Prame-His reference standard of known protein concentration.
After
plotting the PD1/3-Prame-His peaks areas in function of the concentration of
standard solutions, the PD1/3-Prame-His content is deduced from the equation
of
the linear regression.
Table 11: PRAME content
Er En
.6i a
1806
No dialysis (sample stored at -
No spiking A
70 C)
No dialysis (sample left
1639
No spiking
overnight at RT)
CL
(.)
CD L-
5 mM Borate buffer pH 9.8¨
1 649
m CO No spiking
E3.15% sucrose
fa o_
Lci Spiking with CpG15- 5 mM Borate buffer pH 9.8¨
1708
m
2 mers solution up to 3.15% sucrose ¨ 100 pg/ml
E a) 100 pg/ml CpG 15-mers
too
Spiking with CpG24- 5 mM Borate buffer pH 9.8¨
1650
mers solution up to 3.15% sucrose ¨ 100 pg/ml
la 100 pg/ml CpG 24-mers
Spiking with CpG30- 5 mM Borate buffer pH 9.8 ¨
1613
mers solution up to 3.15% sucrose ¨ 100 pg/ml
100 pg/ml CpG 30-mers
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Spiking with PLG- 5 mM Borate buffer pH 9.8¨
1612
24mers solution up to 3.15% sucrose ¨ 100 pg/ml G
100 pg/ml PLG 24-mers
'
'
Spiking with CpG15-
1692
mers solution up to H
100 pg/ml
Spiking with CpG24-
1662
mers solution up to I
mM Borate buffer pH 9.8 ¨
100 pg/ml
3.15% sucrose
Spiking with CpG30-
1634
mers solution up to J
100 pg/ml
Spiking with PLG-
1616
24mers solution up to K
100 pg/ml
_
The Prame content measured in all samples was consistent in all samples. No
significant differences in Prame content could be observed between the sample
in
5 Borate buffer and the samples containing different meres CpG or PLG.
Sarcosyl content by RP-HPLC
A measure of the residual LS content is done after dialyse to ensure that the
LS has
been well removed.
Material & Method: LS content is measured by RP-HPLC technique
- Column: Waters SunFire C18 5pm (4.6x100mm Column)
- UV detector (214nm)
- Flow: 1m1/min
- Temperature: 40 C
- Elution gradient:
o Solvent A: 95% Acetonitrile ¨ 5% H20 ¨ 0.1% TFA
o Solvent B: 5% Acetonitrile ¨ 95% H20 ¨ 0.1% TFA
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Table 12
Phase Time (min) %A
Equilibration 0 50
Gradient 1.4 100
Step 5.6 100
Gradient 5.9 50
Step 9.9 50
Results
Table 13: LS content
7arr-' "" content
2R
3.0
We conclude to an efficient removal of LS for dialysed samples (C to K): all
LS
concentration measured ranges between <0.5pg/m1 and 5.0 pg/ml.
CpG and PLG content by IEX.
The amount of CpG and PLG in all samples was calculated by HPLC using
homologous material as reference standard. It is demonstrated in the table
below
that the poly anions were present at a concentration close to 100 pg/ml as
intended.
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Table 14: PLG and CpG content
PLG content
Diafiltrati on Echantil Ions PLG 24 mers (
g/mL)
spike 100 g PLG 24 mers contre buffer + CpG G 105
spike 100 g PLG 24 mers contre buffer seul K 105
CpG content
Diafiltrati on Echantil Ions CpG g/ml
spike 100 g CpG 15 me rs contre buffer + CpG D 98
spike 100 g CpG 15 me rs contre buffer seul H 100
spike 100 g CpG 24 me rs contre buffer + CpG E 99
spike 100 g CpG 24 me rs contre buffer seul I 95
spike 100 g CpG 30 me rs contre buffer + CpG F 108
spike 100 g CpG 30 me rs contre buffer seul J 107
Size by Dynamic light scattering
Figure 21/21 demonstrates that antigen size is controlled in presence of CpG -
15mers, CpG 24-mers, CpG 30-mers and PLG. CpG's seems to have a very slight
better impact on antigen size stability than PLG (concentration improvement to
consider).
41
